scholarly journals The NSD2 p.E1099K Mutation in Relapse Pediatric Acute Lymphoblastic Leukemia Is Linked to Mercaptopurine Resistance

Blood ◽  
2018 ◽  
Vol 132 (Supplement 1) ◽  
pp. 3962-3962
Author(s):  
Jason Saliba ◽  
Joanna Pierro ◽  
Nikki Ann Evensen ◽  
Anita Qualls ◽  
Natasha Belsky ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Counting ◽  
Type I ◽  
Type Ii ◽  
Set Domain

Abstract Introduction: While the outcome for children with acute lymphoblastic leukemia (ALL) has improved dramatically, the prognosis for those who relapse remains poor. One of the most common alterations found at relapse is the p.E1099K missense change within the SET domain of NSD2, a histone methyltransferase that di-methylates histone 3 lysine 36 (H3K36). NSD2 has 3 isoforms, two of which, Type II (canonical) and REIIBP (C-terminal), contain the SET domain, and another, Type I (N-terminal), that does not. The p.E1099K mutation leads to increased enzymatic activity, but pathways leading to a clonal advantage are unknown in ALL. Methods: We used short hairpin RNAs (shRNAs) to target knockdown of two combinations of NSD2 isoforms: shI/II targets Types I and II, shII/RE targets Type II and REIIBP. Three different B-cell lines (Reh, 697, and KOPN-8) with 2 wildtype (WT) copies of NSD2 were stably transduced with shII/RE. Two B-Cell lines, RS4;11 and RCH-ACV, heterozygous for the NSD2 p.E1099K mutation, were transduced with shI/II and shII/RE. As a control, each B-cell line was stably transduced with a scrambled non-targeting (NT) shRNA. NSD2 knockdown was confirmed by Western Blots. Cell lines were treated for 5 days with chemotherapy agents commonly used in pediatric ALL treatments (mercaptopurine (MP), cytarabine, methotrexate, prednisone, and doxorubicin). Cytotoxicity was assessed by CellTiter- Glo® and significance between IC50s was determined by ANOVA and post hoc Tukey test. Cell proliferation was measured by cell counting with trypan blue. Cell cycle progression in RS4;11 lines was monitored with Edu staining and flow cytometry with and without exposure to MP. Results: Similar to previously reported results, knockdown of NSD2 in the 3 WT B-cell lines had no effect on cell proliferation. However, shI/II reduced growth by 40% in RS4;11 and 20% in RCH-ACV, while shII/RE decreased proliferation by 45% in RS4;11 and 55% in RCH-ACV when compared to their NT control. In RS4;11, both shI/II and shII/RE led to a similar 10% decrease in cells progressing through S phase compared to NT, which could be due to either a slower progression through cell cycle or less cells entering the cell cycle. Knockdown of NSD2 resulted in sensitivity to 6MP compared to NT in both RS4;11 and RCH-ACV lines. RS4;11 shII/RE had an IC50 3.2-fold more sensitive ( p<.01) and the RS4;11 shI/II IC50 was 1.25-fold more sensitive (NS) versus the NT control. Similarly, RCH-ACV shII/RE had an IC50 3.4-fold more sensitive (p<.01) and the RCH-ACV shI/II IC50 was 2.6-fold more sensitive (p<.01) compared to the NT control. No significant changes in drug sensitivity were noted for the 3 WT NSD2 knockdown B-cell lines compared to their NT controls. During a 120 hour exposure to MP, 34% more RS4;11 shII/RE cells were arrested in the G phase than NT controls, while 26% more RS4;11 shI/II cells were arrested in G phase relative to NT controls. This result indicates MP exposure leads to a reduced percentage of knockdown cells able to progress through the cell cycle. Overall, simultaneously reduced expression of Type II and REIIBP had a greater effect of on cell proliferation and MP response compared to the co-reduction of Types I and II NSD2 in the p.E1099K heterozygous cell lines. Conclusion: The p.E1099K mutation confers a growth advantage and resistance to MP, a cornerstone of ALL therapy. Concurrent reduction of Type II and REIIBP expression by shII/RE resulted in the largest impact on proliferation and MP sensitivity. Both of these isoforms include the SET domain containing the p.E1099K mutation, which indicates one or both isoforms could be responsible for changes in the chromatin state and other possible alterations that lead to a clonal advantage. Based on our findings, determining the mechanism of resistance to MP imparted by NSD2 p.E1099K is now a top priority. Disclosures No relevant conflicts of interest to declare.

In Vitro Effects of PI3Kδ Inhibitor GS-1101 (Cal-101) in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3534-3534 ◽  
Author(s):  
Nathalie Y Rosin ◽  
Stefan Koehrer ◽  
Ekaterina Kim ◽  
Susan O'Brien ◽  
William G. Wierda ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Growth ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Lymphocytic Leukemia ◽  
P Value ◽  
B Cell Malignancies ◽  
Growth Experiments

Abstract Abstract 3534 Acute lymphoblastic leukemia (ALL) is a highly heterogeneous disease. B-cell acute lymphoblastic leukemia (B-ALL) is characterized by uncontrolled proliferation of immature lymphoid blasts with suppression of normal hematopoiesis. Phosphoinositide 3-kinases (PI3K) transmit activation signals from diverse transmembrane receptors, leading to generation of phosphatidylinositol- 3,4,5-trisphosphate (PIP3) which promotes proliferation, differentiation, migration, and survival in lymphocytes and various other cell types. A knockout mouse model of the PI3K isoform p110δ demonstrates a unique role of p110δ (PI3Kδ) in B cell receptor (BCR) signaling. This is corroborated by clinical efficacy of the PI3Kδ inhibitor GS-1101 in mature B cell malignancies, especially in chronic lymphocytic leukemia (CLL). In contrast to mature B cell malignancies, expression and function of PI3Kδ in B-ALL has not been well characterized. We therefore analyzed PI3Kδ expression and effects of the PI3Kδ inhibitor GS-1101 in B-ALL. To screen efficacy of GS-1101 in B-ALL subsets, we performed viability and proliferation assays, using a panel of B-ALL cell lines, derived from different B-cell development stages (Pro-B: REH, RS4;11, Nalm-20, Nalm-21, TOM-1; Pre-B: Nalm-6, Kasumi-2, KOPN-8, SMS-SB, RCH-ACV, 697; Mature: Tanoue, Ball-1 unknown: CCRF-SB). A key downstream effector of PI3K is the serine/threonine kinase Akt, whose phosphorylation is used as a common readout of PI3K activation status. Western Blot analysis of the 15 cell lines showed almost identical levels of phospho-Akt (Ser473) in all tested cell lines, suggesting constitutive PI3K activity. To investigate the ability of GS-1101 to inhibit B-ALL cell proliferation, we performed cell growth experiments. Among the pre-B cell lines 4 of 6 showed a marked decrease in proliferation, 2 other pre-B cell lines showed a minor decrease. In contrast, none of the pro-B or mature B-ALL cell lines were affected by GS-1101. To explore the effects of GS-1101 on cell cycle of B-ALL cells, cell lines were treated with GS-1101 at concentrations ranging from 0.5μM to 5μM. In accordance with the cell growth experiments, G1 phase arrest and reduced numbers of S phase cells were detected in pre-B cell lines after GS-1101 treatment, but not in the pro-B or mature B cell lines. Next, we examined GS-1101 effects on metabolism of B-ALL cells via XTT (sodium 2,3,-bis(2-methoxy-4-nitro-5-sulfophenyl)-5-[(phenylamino)-carbonyl]-2H-tetrazolium inner salt) staining. Cell lines were treated with GS-1101 concentrations between 0.1μM and 5μM for 3 days prior to XTT measurement. Pre-B cells showed a significant (p-value <0.0001) decrease in normalized absorbance compared to the control (without treatment) indicating a decrease in cellular viability. Finally, preliminary co-culture experiments of primary B-ALL samples and KUSA-H1 bone marrow stromal cells revealed significantly reduced B-ALL cell viability after GS-1101 treatment, signifying that GS-1101 can overcome microenviromental-mediated B-ALL cell protection; this is similar to that in other B cell malignances. In summary, these experiments demonstrate that GS-1101 inhibits growth, cell cycle progression and metabolic activity of pre-B ALL cells. Validation of these data with primary patient samples is ongoing. Disclosures: Lannutti: Gilead Sciences Inc: Employment.

The Role of MXD3 in Human Precursor B Cell Acute Lymphoblastic Leukemia

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3523-3523
Author(s):  
Gustavo Barisone ◽  
Noriko Satake ◽  
Carly Lewis ◽  
Kit Lam ◽  
Jan A. Nolta ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cell Lines ◽  
Viral Vector ◽  
Lymphoblastic Leukemia ◽  
Mouse Bone Marrow ◽  
Normal Cells ◽  
Hh Signaling

Abstract Abstract 3523 Disregulation of the Hedgehog (Hh) signaling pathway is known to drive proliferation in several cancers, including hematopoietic malignancies. The role of the Hh pathway in precursor B cell (preB) acute lymphoblastic leukemia (ALL), the most common leukemia in children, is unclear; however, recent reports suggest that Hh signaling is involved in the development of B cell precursors as well as the self-renewal and survival of B cell malignancies. MXD3 is a transcription factor previously shown in our lab to be a novel member of the Hh pathway (Yun, Rust, Ishimaru, Diaz, Mol Bio Cell, 2007). We have previously shown that MXD3 is expressed in medulloblastoma (the most common brain tumor in children) and that its knockdown reduces proliferation of human medulloblastoma cell lines (Barisone et al., PLoS, 2012). In the current study, we investigated a possible role for MXD3 in preB ALL cell proliferation. Using quantitative real-time reverse-transcription PCR (qRT-PCR) we observed high levels of MXD3 mRNA expression in 8 primary preB ALL samples as well as in the preB ALL cell lines Reh and JM1. However, MXD3 levels were very low in mobilized peripheral blood mononuclear cells from healthy donors, mouse bone marrow and spleen. Indeed, MXD3 levels in the primary ALL cells and cell lines ranged between 13 and 35 fold increase when compared to the normal cells. Immunoblot analysis with anti-MXD3 monoclonal antibodies confirmed that the protein was present in the ALL samples but not in normal cells. We investigated the role of MXD3 in cell proliferation and survival, by silencing MXD3 in the Reh cell line. We used lentiviral delivery to knockdown MXD3 using an RNA interference approach. Briefly, lentiviruses were produced carrying a short hairpin RNA sequence specific for MXD3 (shMXD3) or a negative control sequence (shCTRL). Upon transduction by the viral vector, MXD3 knockdown was confirmed at both the RNA and protein level. Within 48 hours after transduction, MXD3 protein levels were reduced >90% in cells infected with the shMXD3 virus but not with the control virus. Interestingly, MXD3 knockdown resulted in decreased proliferation in Reh cells, supporting our hypothesis that it may be involved in the maintenance of this malignancy. As a first step toward understanding the mechanisms by which MXD3 exerts its function in leukemia cells, we analyzed cell cycle progression and apoptosis levels after knockdown using flow cytometry. We observed no significant differences in the G0/G1, S or G2/M populations between experimental and control samples. However, samples where MXD3 had been knocked down showed higher levels of apoptosis when compared to controls. Taken together, our results suggest that MXD3 is important for preB ALL cell proliferation, possibly by acting as an anti-apoptotic factor. Therefore, MXD3 may represent a suitable candidate for future efforts in developing targeted therapies against preB ALL. Disclosures: No relevant conflicts of interest to declare.

scholarly journals miR-153-3p Suppresses Inhibitor of Growth Protein 2 Expression to Function as Tumor Suppressor in Acute Lymphoblastic Leukemia

2019 ◽  
Vol 18 ◽  
pp. 153303381985299 ◽  
Author(s):  
Jian Jiang ◽  
Yan Liu ◽  
Yanxia Zhao ◽  
Fei Tian ◽  
Gaoyan Wang
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Leukemia Cell ◽  
Cell Counting ◽  
Migration And Invasion ◽  
Western Blot Assay ◽  
Leukemia Cell Lines ◽  
Direct Target

Alterations in microRNAs expression can accelerate the development of human cancers. However, the role of miR-153-3p in acute lymphoblastic leukemia remains unknown. The expression of miR-153-3p in acute lymphoblastic leukemia cell lines was measured by quantitative real-time polymerase chain reaction. Effects of miR-153-3p expression on acute lymphoblastic leukemia cell proliferation, migration, and invasion were examined by Cell Counting Kit-8 assay, wound healing assay, and Transwell invasion assay, respectively. We then validated inhibitor of growth protein 2 as a direct target of miR-153-3p through bioinformatics analysis, luciferase activity reporter assay, and Western blot assay. The miR-153-3p expression was decreased in acute lymphoblastic leukemia cell lines. Cell proliferation, migration, and invasion of acute lymphoblastic leukemia were obviously decreased by miR-153-3p overexpression. Moreover, inhibitor of growth protein 2 was validated as a direct target of miR-153-3p and the overexpression of inhibitor of growth protein 2 reversed the suppressive effects of miR-153-3p on acute lymphoblastic leukemia cell behaviors. Based on these results, we provided evidence that miR-153-3p might be a target for the treatment of acute lymphoblastic leukemia.

Efficacy of PRT060318, a Novel Highly Specific SYK Inhibitor, in Acute Lymphoblastic Leukemia (ALL)

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 3532-3532
Author(s):  
Stefan KÖhrer ◽  
Greg Coffey ◽  
Ekaterina Kim ◽  
Nathalie Y Rosin ◽  
Uma Sinha ◽  
...  
Keyword(s):  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
B Cells ◽  
B Cell ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Cell Phenotype ◽  
Bcr Signaling ◽  
Syk Inhibitor ◽  
Induction Of Apoptosis

Abstract Abstract 3532 B cell acute lymphoblastic leukemia (B-ALL) arises by transformation of progenitor (pre-B) cells. Cure rates in adults remain low and treatment is complicated by microenvironment-mediated resistance to cytotoxic drugs, indicating an urgent need for development of new, more targeted treatment approaches. Spleen tyrosine kinase (Syk), a B cell receptor (BCR)-associated tyrosine kinase, recently was identified as a novel therapeutic target in mature B cell malignancies, such as chronic lymphocytic leukemia (CLL). Besides its role in BCR signaling in mature B cells, Syk also plays an important role in maintenance and expansion of immature B cells. Syk-deficient mice display a severe defect of B lymphopoiesis, with a block at the pro-B to pre-B transition, consistent with a key role for Syk in pre-BCR signaling. We therefore hypothesized that pre-B ALL cells which express pre-BCRs might be more sensitive to Syk inhibition than other ALL subtypes, which either do not have functional pre-BCRs (pro-B ALL) or display modifying genetic lesions (Ph+/BCR-ABL+ B-ALL). PRT060318, a highly specific, ATP-competitive, small molecular inhibitor of Syk, has preclinical activity in CLL and DLBCL models (Hoellenriegel J et al., Leukemia 26:1576–83, 2012). Syk specificity was confirmed by cellular and non-cellular kinase inhibition assays. We performed metabolic assays using the tetrazolium dye XTT to screen B-ALL cell lines (RS4;11, REH, TOM-1, Nalm-20, Nalm-21, Nalm-6, 697, Kasumi-2, KOPN-8, RCH-ACV, SMS-SB) for responses to PRT060318. ALL cells were incubated with increasing concentrations of PRT060318 (100 nM – 10 μM) for 72 hours. Based on the XTT assays, we determined half maximal inhibitory concentration (IC50), and separated B-ALL cells into responsive (IC50 < 4.5 μM) and non-responsive (IC50 > 4.5 μM) groups. Interestingly, the responsive group consisted only of pre-B ALL cells (CD10+, TdT+, cytoIgμ+), whereas the resistant group comprised only ALL cell lines with a pro-B cell phenotype (CD10+/−, TdT+, cytoIgμ-). All BCR-ABL positive cell lines tested exhibit the pro-B cell phenotype and, similar to their BCR-ABL negative counterparts, were resistant to PRT060318. The Figure depicts XTT assay results for two PRT060318-sensitive pre-B (grey lines) and two resistant pro-B (black lines) B-ALL cell lines. Western Blot analysis demonstrated SYK protein expression in all B-ALL cell lines, excluding lack of target protein expression as possible cause for different response rates between pre-B and pro-B ALL cells. Cell proliferation assays revealed a significant, dose-dependent inhibition of pre-B ALL cell proliferation by PRT060318. Concordantly, dose depended S phase reduction was detected in all PRT060318-sensitive cell lines. In apoptosis assays, in which sensitive pre-B ALL lines were incubated with increasing concentrations of PRT060318 (up to 5 μM) we did not find any significant induction of apoptosis, suggesting anti-proliferative, rather than cytotoxic effects as a main mechanism of action of PRT060318. Preliminary data of primary ALL patient samples, cultured with KUSA-H1 marrow stroma cells in the presence or absence of PRT060318 demonstrate induction of apoptosis in 3 out of 10 samples, other assays are ongoing. In conclusion, we provide evidence that the Syk inhibitor PRT060318 thwarts pre-B ALL cell proliferation, providing a first rationale for clinical testing of PRT060318 in selected patients with B cell ALL. Disclosures: Coffey: Portola Pharmaceuticals Inc.: Employment. Sinha:Portola Pharmaceuticals Inc.: Employment. Pandey:Portola Pharmaceuticals Inc.: Employment.

Restraining the Proliferation of Acute Lymphoblastic Leukemia Cells by Genistein through Up-regulation of B-cell Translocation Gene-3 at Transcription Level

2019 ◽  
Vol 8 ◽  
Author(s):  
Masoumeh Abedi Nejad ◽  
Mohsen Nikbakht ◽  
Masoomeh Afsa ◽  
Kianoosh Malekzadeh
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
B Cell ◽  
Cancer Cells ◽  
Cell Lines ◽  
Lymphoblastic Leukemia ◽  
Suppressor Gene ◽  
Transcription Level ◽  
Hematopoietic Malignancy ◽  
Proliferative Effect ◽  
Cancer Agent

Background: Acute lymphoblastic leukemia (ALL) is a highly prevalent pediatric cancer accounting for approximately 78% of leukemia cases in patients younger than 15 years old. Different studies have demonstrated that B-cell translocation gene 3 (BTG3) plays a suppressive role in the progress of different cancers. Genistein is considered a natural and biocompatible compound and a new anti-cancer agent. In this study, we evaluate the effect of genistein on BTG3 expression and proliferation of ALL cancer cells. Materials and Methods: ALL cell lines (MOLT4, MOLT17, and JURKAT) were cultured in standard conditions. Cytotoxicity of genistein was detected using MTT assay. The cells were treated with different concentrations of genistein (10, 25, 40, and 55μM) for 24, 48, and 72 hours, and then cell viability and growth rate were measured. The quantitative real-time polymerase chain reaction was applied to investigate the effect of genistein on BTG3 expression. Results: The percentage of vital cells treated with genistein significantly decreased compared to the non-treated cells, showed an inverse relationship with an increasing genistein concentration. The present study suggests a dose of 40μM for genistein as a potent anticancer effect. Genistein could elevate BTG3 for 1.7 folds in MOLT4 and JURKAT and 2.7 folds in MOLT17 cell lines at transcription level conveged with 60 to 90% reduction in the proliferation rate of cancer cells. Conclusion: Up-regulation of BTG3 as a tumor suppressor gene can be induced by genistein. It seems that BTG3 reactivation can be introduced as another mechanism of anti-proliferative effect of genistein and could be considered as a retardant agent candidate against hematopoietic malignancy.[GMJ. 2019;inpress:e1229]

scholarly journals BRD4 PROTAC degrader ARV-825 inhibits T-cell acute lymphoblastic leukemia by targeting 'Undruggable' Myc-pathway genes

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Shuiyan Wu ◽  
You Jiang ◽  
Yi Hong ◽  
Xinran Chu ◽  
Zimu Zhang ◽  
...  
Keyword(s):  
Cell Cycle ◽  
Cell Proliferation ◽  
Acute Lymphoblastic Leukemia ◽  
Caspase 3 ◽  
Lymphoblastic Leukemia ◽  
Rna Seq ◽  
Cell Acute Lymphoblastic Leukemia ◽  
Bet Protein

Abstract Background T-cell acute lymphoblastic leukemia (T-ALL) is an aggressive disease with a high risk of induction failure and poor outcomes, with relapse due to drug resistance. Recent studies show that bromodomains and extra-terminal (BET) protein inhibitors are promising anti-cancer agents. ARV-825, comprising a BET inhibitor conjugated with cereblon ligand, was recently developed to attenuate the growth of multiple tumors in vitro and in vivo. However, the functional and molecular mechanisms of ARV-825 in T-ALL remain unclear. This study aimed to investigate the therapeutic efficacy and potential mechanism of ARV-825 in T-ALL. Methods Expression of the BRD4 were determined in pediatric T-ALL samples and differential gene expression after ARV-825 treatment was explored by RNA-seq and quantitative reverse transcription-polymerase chain reaction. T-ALL cell viability was measured by CCK8 assay after ARV-825 administration. Cell cycle was analyzed by propidium iodide (PI) staining and apoptosis was assessed by Annexin V/PI staining. BRD4, BRD3 and BRD2 proteins were detected by western blot in cells treated with ARV-825. The effect of ARV-825 on T-ALL cells was analyzed in vivo. The functional and molecular pathways involved in ARV-825 treatment of T-ALL were verified by western blot and chromatin immunoprecipitation (ChIP). Results BRD4 expression was higher in pediatric T-ALL samples compared with T-cells from healthy donors. High BRD4 expression indicated a poor outcome. ARV-825 suppressed cell proliferation in vitro by arresting the cell cycle and inducing apoptosis, with elevated poly-ADP ribose polymerase and cleaved caspase 3. BRD4, BRD3, and BRD2 were degraded in line with reduced cereblon expression in T-ALL cells. ARV-825 had a lower IC50 in T-ALL cells compared with JQ1, dBET1 and OTX015. ARV-825 perturbed the H3K27Ac-Myc pathway and reduced c-Myc protein levels in T-ALL cells according to RNA-seq and ChIP. In the T-ALL xenograft model, ARV-825 significantly reduced tumor growth and led to the dysregulation of Ki67 and cleaved caspase 3. Moreover, ARV-825 inhibited cell proliferation by depleting BET and c-Myc proteins in vitro and in vivo. Conclusions BRD4 indicates a poor prognosis in T-ALL. The BRD4 degrader ARV-825 can effectively suppress the proliferation and promote apoptosis of T-ALL cells via BET protein depletion and c-Myc inhibition, thus providing a new strategy for the treatment of T-ALL.

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